GTF file error when using easyRNAseqâ

Natalia [guest] guest at bioconductor.org
Mon Nov 11 16:31:59 CET 2013


Dear all,
I would like to make a count table to use it in DESeq. I´ve tried to use easyRNAseq but I have a problem with the annotation file. I’ve downloaded the file Gallus_gallus.Galgal4.73.gtf from Ensembl. As I run into the problem Error in .doBasicCount(obj) : The genomicAnnotation slot is empty, I modified the file and added chr before the chromosome number. The next problem was this:

Your gtf file: Gallus_gallus.Galgal4.73.gtf does not contain all the required fields: gene_id, transcript_id, exon_number, gene_name.

To solve this problem:
- I deleted all the entries without gene_name (first example):

gene_id "ENSGALG00000009771"; transcript_id "ENSGALT00000015891"; exon_number "1"; gene_biotype "protein_coding"; exon_id "ENSGALE00000301221";

gene_id "ENSGALG00000009783"; transcript_id "ENSGALT00000015914"; exon_number "2"; gene_name "GOLGB1"; gene_biotype "protein_coding"; transcript_name "GOLGB1-201"; exon_id "ENSGALE00000105891";

- I checked the chromosome numbers and deleted the entries that didn’t match any chromosome from BSgenome.Ggallus.UCSC.galGal4 (I can’t find any entry corresponding to chr32 in the Gallus_gallus.Galgal4.73.gtf file, I don’t know if it is a problem):

- I searched for semicolons and single quotes ‘ in the gene names, but I didn’t find any on the final file.

- I deleted all the columns after gene_name.

So finally the annotation file entries look like this:
chr1	protein_coding	exon	19962541	19963992
	.	+	.	gene_id "ENSGALG00000000003"; transcript_id "ENSGALT00000000003"; exon_number "2"; gene_name "PANX2";

Nothing works; the error message is always the same. So, I don’t know what else I can do. Could you please help me?
Thank you in advance!
Cheers

Natalia


here is my code:
> count.table <- easyRNASeq("/RNAseqGallus", organism="Ggallus", chrSizes="chrSizes", annotationMethod="gtf", annotationFile="Gallus_gallus.Galgal4.73.gtf", count="genes", summarization="geneModels", format="bam", gapped=TRUE, filenames=c("NS1gallus.bam","NS2gallus.bam"), outputFormat="DESeq", conditions=conditions)
Checking arguments... 
Fetching annotations... 
Read 334620 records
Error en .getGtfRange(organismName(obj), filename = filename, ignoreWarnings = ignoreWarnings,  : 
  Your gtf file: Gallus_gallus.Galgal4.73.gtf does not contain all the required fields: gene_id, transcript_id, exon_number, gene_name.
Además: Mensajes de aviso perdidos
1: In easyRNASeq("/RNAseqGallus", organism = "Ggallus", chrSizes = "chrSizes",  :
  Your organism has no mapping defined to perform the validity check for the UCSC compliance of the chromosome name.
Defined organism's mapping can be listed using the 'knownOrganisms' function.
To benefit from the validity check, you can provide a 'chr.map' to your 'easyRNASeq' function call.
As you did not do so, 'validity.check' is turned off
2: In .Method(..., deparse.level = deparse.level) :
  number of columns of result is not a multiple of vector length (arg 1)

> traceback()
6: stop(paste("Your gtf file: ", filename, " does not contain all the required fields: ", 
       paste(fields, collapse = ", "), ".", sep = ""))
5: .getGtfRange(organismName(obj), filename = filename, ignoreWarnings = ignoreWarnings, 
       ...)
4: fetchAnnotation(obj, method = annotationMethod, filename = annotationFile, 
       ignoreWarnings = ignoreWarnings, ...)
3: fetchAnnotation(obj, method = annotationMethod, filename = annotationFile, 
       ignoreWarnings = ignoreWarnings, ...)
2: easyRNASeq("/RNAseqGallus", organism = "Ggallus", chrSizes = "chrSizes", 
       annotationMethod = "gtf", annotationFile = " Gallus_gallus.Galgal4.73.gtf ", 
       count = "genes", summarization = "geneModels", format = "bam", 
       gapped = TRUE, filenames = c("NS1gallus.bam", "NS2gallus.bam"), 
       outputFormat = "DESeq", conditions = conditions)
1: easyRNASeq("/RNAseqGallus", organism = "Ggallus", chrSizes = "chrSizes", 
       annotationMethod = "gtf", annotationFile = " Gallus_gallus.Galgal4.73.gtf ", 
       count = "genes", summarization = "geneModels", format = "bam", 
       gapped = TRUE, filenames = c("NS1gallus.bam", "NS2gallus.bam"), 
       outputFormat = "DESeq", conditions = conditions)


 -- output of sessionInfo(): 

> sessionInfo()
R version 3.0.2 (2013-09-25)
Platform: x86_64-w64-mingw32/x64 (64-bit)

locale:
[1] LC_COLLATE=Spanish_Spain.1252  LC_CTYPE=Spanish_Spain.1252    LC_MONETARY=Spanish_Spain.1252 LC_NUMERIC=C                   LC_TIME=Spanish_Spain.1252    

attached base packages:
[1] parallel  stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] BSgenome.Ggallus.UCSC.galGal4_1.3.18 BSgenome_1.30.0                      easyRNASeq_1.8.1                     ShortRead_1.20.0                    
 [5] Rsamtools_1.14.1                     GenomicRanges_1.14.3                 DESeq_1.14.0                         lattice_0.20-23                     
 [9] locfit_1.5-9.1                       Biostrings_2.30.0                    XVector_0.2.0                        IRanges_1.20.4                      
[13] edgeR_3.4.0                          limma_3.18.2                         biomaRt_2.18.0                       Biobase_2.22.0                      
[17] genomeIntervals_1.18.0               BiocGenerics_0.8.0                   intervals_0.14.0                     BiocInstaller_1.12.0                

loaded via a namespace (and not attached):
 [1] annotate_1.40.0      AnnotationDbi_1.24.0 bitops_1.0-6         DBI_0.2-7            genefilter_1.44.0    geneplotter_1.40.0   grid_3.0.2          
 [8] hwriter_1.3          latticeExtra_0.6-26  LSD_2.5              RColorBrewer_1.0-5   RCurl_1.95-4.1       RSQLite_0.11.4       splines_3.0.2       
[15] stats4_3.0.2         survival_2.37-4      tools_3.0.2          XML_3.98-1.1         xtable_1.7-1         zlibbioc_1.8.0  


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